gst usp7 recombinant protein  (Sino Biological)

Journal: The Journal of Biological Chemistry

Article Title: HMGN1 and HMGN2 are recruited to acetylated and histone variant H2A.Z-containing nucleosomes to regulate chromatin state and transcription

doi: 10.1016/j.jbc.2025.110997

Figure Lengend Snippet: HMGN1 and HMGN2 reduce p300-mediated acetylation of the H3 tail . A , Western blot analysis of HAT reaction mixtures containing equal amounts of recombinant mononucleosomes (canonical nuc.) with 147 base pairs of 601 sequence DNA, preincubated with variable amounts of recombinant GST-HMGN1 or GST-HMGN1ΔC protein and then incubated with equal amounts of recombinant p300 and acetyl-CoA. H3 lysine acetylation of K18, K23, and K27 was imaged via PTM-specific antibodies. B , Western blot analysis of HAT reaction mixtures containing equal amounts of recombinant mononucleosomes (canonical nuc.) with 147 base pairs of 601 sequence DNA, preincubated with variable amounts of recombinant GST-HMGN2 or GST-HMGN2ΔC protein and then incubated with equal amounts of recombinant p300 and acetyl-CoA. H3 lysine acetylation of K18, K23, and K27 was imaged via PTM-specific antibodies. C , Western blot analysis of HAT reaction mixtures containing equal amounts of recombinant mononucleosomes (canonical nuc.) or recombinant H2A.Z-containing mononucleosomes with 147 base pairs of 601 sequence DNA, preincubated with variable amounts of recombinant GST-HMGN1 protein and then incubated with equal amounts of recombinant p300 and acetyl-CoA. H3 lysine acetylation of K27 was imaged via PTM-specific antibodies. D , Western blot analysis of HAT reaction mixtures containing equal amounts of recombinant mononucleosomes (canonical nuc.) or recombinant H2A.Z-containing mononucleosomes with 147 base pairs of 601 sequence DNA, preincubated with variable amounts of recombinant GST-HMGN2 protein and then incubated with equal amounts of recombinant p300 and acetyl-CoA. H3 lysine acetylation of K27 was imaged via PTM-specific antibodies. E , Western blot analysis of HAT reaction mixtures containing equal amounts of recombinant mononucleosomes (canonical nuc.) or recombinant H2AE61A mononucleosomes with 147 base pairs of 601 sequence DNA, preincubated with variable amounts of recombinant GST-HMGN1 protein and then incubated with equal amounts of recombinant p300 and acetyl-CoA. H3 lysine acetylation of K27 was imaged via PTM-specific antibodies. F , Western blot analysis of HAT reaction mixtures containing equal amounts of recombinant mononucleosomes (canonical nuc.) or recombinant H2AE61A mononucleosomes with 147 base pairs of 601 sequence DNA, preincubated with variable amounts of recombinant GST-HMGN2 protein and then incubated with equal amounts of recombinant p300 and acetyl-CoA. H3 lysine acetylation of K27 was imaged via PTM-specific antibodies. GST, glutathione- S -transferase; HAT, histone acetyltransferase; HMGN, High Mobility Nucleosome-binding protein; PTM, post-translational modification.

Article Snippet: HAT assays were performed at 37 ̊C for 30 min with 500 nM purified p300 (BPS Biosciences; catalog no.: 50071), 300 nM nucleosome substrate (Epicypher 16-0009, 16-1014, or 16-1014), 100 μM acetyl-CoA (Sigma; catalog no.: A2056), and 100 nM, 300 nM, or 900 nM purified GST-HMGN protein (HMGN1, HMGN2, HMGN1ΔC, or HMGN2ΔC) in a reaction volume of 10 μl (in HAT buffer: 50 nM Tris–HCl [pH 8.0], 10% glycerol, 1 mM DTT, 0.1 mM EDTA, 1 mM PMSF, and 10 mM sodium butyrate).

Techniques: Western Blot, Recombinant, Sequencing, Incubation, Binding Assay, Modification